In recent years the quest for a safe blood substitute has accelerated rapidly. The demand often exceeds the supplies of blood available from human donors. In addition, in many parts of the world, the whole blood supply can be hazardous. Therefore, there is a need to develop blood substitutes.
One of the most important functions of blood is to carry oxygen from lungs to support tissue respiration. Hemoglobin (Hb) is an attractive oxygen carrier in the development of a clinical blood substitute, given its attributes as a respiratory pigment of extensive solubility, uptake and release of oxygen, and above all its capability of transporting a large quantity of oxygen. However, one fundamental disadvantage of free Hb itself as a blood substitute arises from its relatively small molecular size of 64.5 kD and consequent high renal excretion rate (EXC) thereby leading to a rapid clearance from the circulation. Therefore, covalent conjugation to carrier polymers has been applied in order to prevent renal excretion of Hb and to prolong its plasma half-life. Such conjugates are referred to as hemoglobin based oxygen carriers (HBOC). Examples of such polymers include: dextran and biopolymer derivatives of dextran, inulin, hydroxyethylstarch, polyethylene glycol, polyvinylpyrrolidone (S.P.Tsai and J. T.-F. Wong, Dextran-Hemoglobin, in: Winslow, R. N., Vandegriff, K. D., and Intaglietta, M. [eds.], 1997 Advances in Blood Substitutes: Industrial Opportunities and Medical Challenges, Birkhauser, Boston; the contents of these references are incorporated herein by reference).
The efficiency of oxygen delivery is determined by the total blood flow and volume, oxygen content, red cell or hemoglobin mass, oxygen affinity and the rate of oxygen consumption. The relationships between oxygen content, delivery and utilization are best exemplified by the Fick's equation. It is therefore apparent that a blood substitute which can carry and deliver a maximal amount of oxygen per unit volume, while maintaining excellent rheologic characteristics, would be ideal.
Dextran-hemoglobin (DxHb) is one of the conjugates that has been proposed as a blood substitute. The combination of water solubility, availability in a wide range of molecular sizes, and lack of significant toxicity or tissue tropism, renders dextran an excellent drug carrier among biodegradable polymers. Covalent conjugation of dextran (Dx) to hemoglobin (Hb) increases the effective size of Hb and, therefore, reduces its excretion rate (EXC) through the renal system. Methods to stabilize the viscosity of the conjugate solution have been proposed, and exchange transfusions with DxHb in dogs and macaques performed (Tam et al, 1976, Tam et al, 1978, Wong 1988). However, hitherto the flow properties of the conjugate have not been investigated.
Examples of DxHb conjugates are provided in U.S. Pat. Nos. 4,064,118 and 4,650,786, the contents of which are incorporated herein by reference. The '118 patent teaches a composition useful as a blood substitute or blood extender which is prepared by chemically coupling hemoglobin (Hb) with dextran (Dx) having a molecular weight of from about 5 kD to 2000 kD. The molecular weight of this DxHb conjugate is in the range of 70-2000 kD. It has however been found that, as compared to hemoglobin, the products according to U.S. Pat. No. 4,064,118 tend to show a somewhat greater affinity for oxygen, but retain the essential oxygen transporting and releasing capability of hemoglobin.
U.S. Pat. No. 4,650,786 describes a modified dextran-hemoglobin complex having reduced oxygen affinity. The molecular weight of this DxHb complex is in the range of 70-2000 kD.
One of the problems associated with DxHb complexes, or modified DxHb complexes, is that the viscosity of the conjugate solution increases on storage thereby rendering the solution unsuitable for administration. The solution to the above problem is described in U.S. Pat. No. 4,900,816, the contents of which are incorporated herein by reference. It has been shown that the activated sites on the dextran moiety may be blocked, and the viscosity on storage stabililized, without affecting the oxygen transport properties of the hemoglobin complex. U.S. Pat. No. 4,900,816 teaches a compound having a molecular weight from about 70 kD to about 200 kD, comprising a hemoglobin residue, an oxygen affinity reducing ligand, a polysaccharide (e.g. dextran), covalently bonded chemical bridging groups and a blocked activating group.
As indicated above, one of the important characteristics of a blood substitute is its rheological properties—in order for the substitute to be physiologically acceptable, its viscosity must not be so high as to hinder flow of blood. Although aggregation of red blood cells is one of the important causes of increased blood viscosity, especially at lower shear rates, the actual mechanism of red cells aggregation is still not completely elucidated. Aggregation of red cells can be brought about by various means. In general, both the viscosity and the RBC aggregation increase with increasing concentration of immunoglobulin; however, the exact relationship between the two appears to be quite complex. It is therefore important to characterize the possible physico-chemical properties of DxHb in its development as blood substitute.
As described below, in the present invention, various preparations of DxHb were synthesized and the rheologic properties of these solutions were examined by measuring their viscosities and their aggregating tendencies which were assessed based on erythrocyte sedimentation rate (ESR) test (Dintenfass 1985, the contents of which are incorporated herein by reference). The presence of macromolecules over a certain “critical concentration” in plasma could induce red blood cell (RBC) aggregation and in turn blood viscosity particularly at low shear rate.
Blood flow in low shear regions, especially in the venous circulation, is greatly reduced by the enhancement of erythrocyte aggregation which increases blood viscosity and impedes capillary flow through sludge formation (Dintenfass 1981). Red blood cell, RBC, aggregation is the result of bridging by macromolecules between adjacent erythrocyte surfaces. When a non-encapsulated hemoglobin-based blood substitute is infused into the circulation, these macromolecules could also interact with erythrocytes and induce RBC aggregation, with ESR being one of the foremost blood rheological parameters to be influenced by such aggregation. Therefore, it is of fundamental interest to examine the ESR enhancement effects of hemoglobin-based blood substitutes in the course of their design and development.
It is known that molecular size is one of the critical determinants of ESR enhancement through the macromolecular bridging mechanism. Dextrans of 20 kD do not induce RBC aggregation and hence, no ESR elevation, but dextrans larger than 40 kD are entirely capable of enhancing ESR (Chien and Jan 1973; the contents of which are incorporated herein by reference). Previous studies showed that macromolecular polymerized hemoglobin larger than 220 kD would induce RBC aggregation, which may increase low-shear-rate blood viscosity and affect the RBC distribution in the circulation (Tsai and Wong 1996; the contents of which are incorporated herein by reference).
The present invention provides a DxHb conjugate having a molecular weight range that results in low EXC and ESR levels and, therefore, provides an effective blood substitute or plasma expander.